Generation and Characterization of a scFv Antibody Against T3SS Needle of Vibrio parahaemolyticus

Abstract

Vibrio parahaemolyticus, a halophilic gramnegative bacterium, is a food-borne pathogen that largely inhabits marine and estuarine environments, and poses a serious threat to human and animal health all over the world. The hollow ''needle'' channel, a specific assemble of T3SS which exists in most of gram-negative bacteria, plays a key role in the transition of virulence effectors to host cells. In this study, needle protein VP1694 was successfully expressed and purified, and the fusion protein Trx-VP1694 was used to immunize Balb/c mice. Subsequently, a phage single-chain fragment variable antibody (scFv) library was constructed, and a specific scFv against VP1694 named scFv-FA7 was screened by phage display panning. To further identify the characters of scFv, the soluble expression vector pACYC-scFv-skp was constructed and the soluble scFv was purified by Ni 2? affinity chromatography. ELISA analysis showed that the scFv-FA7 was specific to VP1694 antigen, and its affinity constant was 1.07 9 10 8 L/mol. These results offer a molecular basis to prevent and cure diseases by scFv, and also provide a new strategy for further research on virulence mechanism of T3SS in V. parahaemolyticus by scFv.

The pathogenesis, detection, and prevention of Vibrio parahaemolyticus

“…The needle subunit may be a useful target protein for screening an effective antibody or inhibitor that can prevent the formation of the needle complex ( Davis and Mecsas, 2007 ). A specific and high-affinity scFv antibody directed against VP1694 (needle subunit) has been prepared, and may play an important role in inhibiting the assembly of T3SS ( Wang et al, 2014b ). Significantly, the above results showed that the specific and functional scFv antibodies against target antigens of V. parahaemolyticus have been prepared successfully, and it provides a solid foundation for the immunological diagnosis and prevention of the diseases caused by V. parahaemolyticus.…”

Multiplicity of Quorum Quenching Enzymes: A Potential Mechanism to Limit Quorum Sensing Bacterial Population

“…Vibrio spp. fight their competitors through toxin loaded molecular guns-the Type III and VI secretion system (T3SS, T6SS) [7,8]. Pseudomonas spp.…”

Control of AHPND by phages: a promising biotechnological approach

“…More recently, Wang et al . () found a specific scFv against VP1694 (a subunit protein of the needle complex of the type III secretion system (T3SS) of V. parahaemolyticus ) named scFv‐FA7. VP1694 immunized female Balb/c mice were used to construct a phage single‐chain fragment variable antibody.…”